The present invention was made with the support of the National Science Foundation Grant No. DIR-8809710. The United States Government may have rights to the invention.
1. Field of the Invention
The present invention relates generally to the support mediums used in electrophoresis. More particularly, the present invention is directed to the use of polymers and copolymers which include hydroxyalkyl esters of acrylic or methacrylic acid or poly(alkylene glycol) esters of methacrylic or acrylic acid as at least part of the support media.
2. Description of Related Art
Support media are commonly used in electrophoresis systems to suppress convection caused by gravity, thermal gradients or concentration gradients. The support media which have been used conventionally include powdered and porous solids, fibrous materials and gels. The powdered and porous materials which are used as electrophoresis media includes cellulose, starch, silica, glass, polyurethane foam and glass powder. For the most part, the powdered and porous solid electrophoresis media have been replaced by gels which have a higher resolving power. The powdered and porous solids are generally reserved for large scale preparative separations.
Fibrous materials, such as paper have been used in electrophoresis for many years. Paper electrophoresis support media became popular due to their low cost and ease of handling. However, the use of paper as a support media has largely been replaced with gels due to problems experienced with variations in different batches of paper and impurities in the paper which caused undesirable and unpredictable absorptive properties.
Cellulose acetate membranes have also been used as an electrophoresis support media. Cellulose acetate membranes do not have the undesirable absorptive properties of paper and have a uniform microporous structure and are chemically inert. However, cellulose acetate must be laminated to a flexible plastic support due to the inherent brittleness of dry cellulose acetate.
The most popular electrophoresis support materials are based on molecular-sieve gels. Starch gels were initially used in the early 1950's for the separation of proteins. However, the narrow range of porosities and the fragile nature of starch gels have rendered them obsolete. Agar and agarose gels have been widely used as an electrophoresis support media. Agar and agarose gels are obtained from polysaccharides extracted from red algae. Agar and agarose gels have not been widely used (except for large DNA molecules) as an electrophoresis support media because of limited sieving properties and a high content of anionic residues, such as sulfate and pyruvate.
Polyacrylamide gel (PAG) has been widely adopted as the support media of choice for the separation of both proteins and DNA. Cross-linked polyacrylamide provides good resolution in many applications because it possesses sieving as well as anti-convective properties. The sieving properties of cross-linked polyacrylamide gels are particularly well-suited for molecular weight separations. By varying the percentage of monomer or cross-linker, the nature of the gel can be changed to suit a variety of separations from small (1,000 kD) peptides to large (500,000 kD) proteins.
Despite the numerous advantages and popularity of polyacrylamide gel support media, there are a number of inconveniences, hazards and limitations which accompany the use of this material. For example, the acrylamide monomer and the bis-acrylamide cross-linker represent a serious health hazard. Although the polymer is not toxic, exposure to the monomer and cross-linker during preparation of the gel poses significant health concerns. In addition, residual and derivative chemicals present during post-electrophoresis processing also pose health concerns.
The health problems associated with acrylamide monomer are compounded by the fact that the toxic effects of acrylamide are cumulative. The toxicity problem associated with acrylamide monomers can be carefully controlled in a research setting. However, toxicity concerns may limit the use of polyacrylamide gel in clinical laboratory settings where people being exposed to the toxins may not be well-informed about the risks associated with polyacrylamide monomer and carefully trained with respect to proper handling procedures.
Another problem associated with polyacrylamide gel support media is the difficulty in forming gels of reproducible properties. Acrylamide monomers and the bis-acrylamide cross-linkers are commercially available as extremely pure and uniform compositions. However, separation of the electrophoresis support media involves a high degree of skill and care. Slight changes in preparation technique from batch to batch results in the formation of gels having slightly different properties. Further, the pouring process for preparation of the gel is prone to minor variations which result in the formation of gels which vary in composition at different locations within the gel.
The variability present in polyacrylamide gel support media results in inconsistent protein migration within a particular gel media. Further, protein migration is not reproducible when different batches of gel are used. As a result of these inconsistencies, polyacrylamide gel support media has not been widely used in clinical applications. Instead, cellulose acetate membranes have been used even though they have considerably less resolution power.
Two dimensional electrophoresis (2DE) is a technique which allows the identification of thousands of molecules simultaneously. In 2DE systems, the samples are subjected to electrophoresis based on two independent variables such as charge and mass. For example, in a first dimension, isoelectric focusing (IEF) is used to separate complex mixtures based on charge. In a second dimension, polyacrylamide gel electrophoresis is used to separate the samples based on mass. The resulting 2-dimensional image contains the positional coordinates and quantity of each species as well as all interconnecting correlations. Unlike a series of one-dimensional separations, the 2DE gel image provides a data base which is suitable for determining individual differences between samples and for the analysis of molecular networks.
The full potential of two-dimensional electrophoresis has been difficult to obtain because of non-uniformities in the polyacrylamide gel support media. For example, the computer matching of up to thousands of protein spots on a two-dimensional electrophoresis is greatly hindered by artifacts in the polyacrylamide gel support media such as bubbles, insoluble material, polymer concentration gradients and cross-link density variabilities. These variabilities or artifacts in the gel give rise to glitches in protein spot structure and gel-to-gel variations in composition that result in irreproducibility of relative protein or DNA migration velocities.
In view of the above problems with present electrophoresis support media, it would be desirable to provide improved support media which overcome the disadvantages set forth above. For example, it would be desirable to provide electrophoresis support media which are non-toxic and easily handled. Further, the procedures and techniques for forming the support media should be simple and easily mastered so that uniform support media with reproducible characteristics can be prepared routinely. Finally, the properties of the support media must be such that they are suitable for use in high performance electrophoresis systems, such as two-dimensional electrophoresis.
Many times it is desirable to use organic solvents in the electrophoresis process. The present electrophoresis medias, such as polyacrylamide gel are not well-suited for use with organic solvents to separate hydrophobic molecules. Accordingly, there is also a need to provide electrophoresis support media which not only have all of the above mentioned desired characteristics, but are also suitable for use with organic solvents.